Coding

Part:BBa_K4808002

Designed by: Zhao Guichun   Group: iGEM23_AIS-China   (2023-10-11)

ilvA*

ilvA* is the mutation of ilvA BBa_K4808001. In E.coli, IIe will have inhibition effect towards the production of threonine dehydratase encoded by ilvA. It will reduce the activity of this enzyme and further reduce the production of a-KB, which is our target product. As a result, we mutated ilvA to eliminate the effect of lle on it.


Characterization

For further improvement on a-kb production levels, we want to optimize threonine's conversion into a-kb, a key step in a-kb production.We tried to use plasmids with different copy numbers to express ilvA to determine the effect on copy number on a-KB production. Thus, we constructed ilvA onto a new plasmid p321-ilvA, which has a low copy number in E. coli (Figure A)

We also mutated ilvA(obtainning ilvA*) to make its expressed enzymes resistant to the inhibition of Isoleucine. Isoleucine is a downstream product of a-kb.Our gene knock out choices do not completely obstruct the pathway continuing downwards of a-kb, thus meaning that Isoleucine is existent in the E.coli cell (Isoleucine inhibits ilvA expressed Threonine Dehydrase, thus negatively affecting Threonine's conversion into a-kb, which is reported in many papers).

We made four base mutations in ilvA (i.e., C1339T, G1341T, C1351G, T1352C), which substitutes the 447th and 451th amino acids (both Leu) into Phe and Ala, respectively (Figure B). After successfully mutating the ilvA gene through PCR with primers coding for the mutated sequences (Figure C), we obtained the strains pw1-ilvA* and p321-ilvA* (ilvA* is the mutated ilvA). The two plasmids, along with pw1-ilvA and p321-ilvA, were transformed into AIS-2, and the a-kb production is measured. The result show that pw1-ilvA* strain was most efficient. Moreover, we found that for both vectors (p321 and pw1), the mutated ilvA* yielded more a-KB compared to the non-mutated ilvA, and no matter whether ilvA is mutated or not, the pw1 plasmid with higher copy number yielded more a-KB than the p321 plasmid with low copy number. 

 star1.png

Figure: (A) the different copy number of plasmid with ilvA. (B) ilvA*'s resistancy to isoleucine inhibition and codon and amino acid differences between the ilvA and the mutated ilvA*. (C) the design for the mutation of ilvA and the sequencing results of ilvA*. (D)the a-kb production of strain AIS-2 with four different plasmid;

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1315
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1315
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1315
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1315
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1315
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1168


References：

Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058

Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036

Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023

Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z

Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959

Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948

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